It has been shown that hepatitis C virus (HCV) populations in vivo are composed of different but highly homologous HCV genomes (quasispecies) as shown in the hypervariable region (HVR) that exists in the N-terminal of the envelope 2 gene of HCV, and that the predominant sequence of the HVR of HCV genomes changes rapidly over time. To further investigate genetic backgrounds of the change in the HVR of HCV genomes, 45 plasma samples serially obtained from nine patients with chronic hepatitis C were studied using population-based analyses. Total RNA was recovered and the envelope gene containing the HVR was amplified by the reverse transcription and nested polymerase chain reaction. The amplified cDNA was examined by the single strand conformation polymorphism (SSCP) analysis. Furthermore, 43 HCV sequences, separated by the SSCP analysis from three patients were determined by the dideoxy chain termination method, and the phylogenetic analysis was performed using the neighbor joining method. The SSCP analysis demonstrated that HCV population within each individual were composed of 1 to 6 quasispecies. These quasispecies populations in vivo changed sequentially in eight of nine patients. Gradual selections of coexisting quasispecies were observed over 6- to 18-month periods in three patients, whereas complete replacements of previous quasispecies by new quasispecies were repeatedly observed over few-month intervals in five patients. The phylogenetic analysis on these quasispecies revealed the continuous accumulation of mutations in two patients and discontinuous appearance of evolutionarily distant quasispecies in one patient. These results indicate that HCV genomes in vivo form quasispecies populations, and that these quasispecies populations change during the natural course of chronic infection. Genetic mechanisms underlining the change of the HVR of HCV genome appear to be either continuous accumulation of mutations or selective overgrowth of preexisting minor variants from the large spectrum of quasispecies populations.